Cell culture method using amino acid-enriched medium

ABSTRACT

Methods of culturing cells capable of producing desired proteins to obtain the proteins by use of a medium from which biological components are excluded as much as possible are provided. Specifically, a culture method characterized by culturing while maintaining a specific amino acid in a culture solution at a high concentration, and a cell culture fed-batch medium for use in the method are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.16/297,086, filed Mar. 8, 2019, which is a Divisional of U.S.application Ser. No. 12/451,003 filed Oct. 22, 2009 which is a U.S.National Stage application of PCT/JP2008/058046, filed on Apr. 25, 2008,which claims priority from Japanese Patent application No. 2007-117426,filed on Apr. 26, 2007, the entire disclosures of all of which areincorporated herein by reference.

The instant application contains a Sequence Listing which has beensubmitted in XML format via Patent Center and is hereby incorporated byreference in its entirety. Said XML copy, created on Apr. 18, 2023, isnamed SequenceListing.xml and is 5,470 bytes.

TECHNICAL FIELD

The present invention relates to methods of culturing cells capable ofproducing desired proteins to obtain the proteins, and media for use inthe methods. In particular, the present invention relates to methods ofculturing cells capable of producing desired proteins to obtain thedesired proteins and to methods of producing proteins by a serum-freeculture method, characterized in that serine in a liquid culture ismaintained at a high concentration. The present invention furtherrelates to cell culture fed-batch media for use in the methods.

BACKGROUND ART

When a natural protein produced by an animal cell was to be obtained byculturing the animal cell, or when a desired protein or the like was tobe prepared by culturing an animal cell transformed with a gene encodingthe desired protein, the culture medium had to be supplemented in therange of 5-20% with an extract derived from a mammal, specifically aserum such as fetal calf serum, for the growth of the animal cell, inaddition to basic nutrients such as salts, sugars, amino acids, andvitamins. The sera derived from mammals, however, entailed disadvantagesthat such mammal-derived sera account for 75-95% of the cost of media,and that stable growth cannot be achieved due to variation in qualityamong lots. Furthermore, since sera derived from mammals cannot besterilized by an autoclave or the like, there is a possibility ofcontamination with viruses or mycoplasmas; although many of them areharmless, they may become additional unknown factors from the viewpointof stable production.

In recent years, there has been great concern that components derivedfrom mammals may be associated with mad cow disease, or BovineSpongiform Encephalopathy (BSE), Transmissible Spongiform Encephalopathy(TSE), and, furthermore, Creutzfeld-Jakob Disease (CJD), and in view ofsafety, an animal cell culture medium free from mammalian components hasbeen sought. Further, a serum contains at least 500 types of proteins,and this complicates isolation/purification of a desired protein as abio-product from a cell culture medium.

To solve the above problems, serum-free culture methods suitable forculturing animal cells in the absence of a serum have been developed. Inthe development of serum-free culture methods, serum-free liquid mediacontaining, as substitutes for the effect of sera, plasma proteins suchas fetuin, transferrin, and albumin, hormones such as steroid hormoneand insulin, growth factors, and nutrient factors such as amino acidsand vitamins have been provided.

Fetuin, insulin, transferrin, and growth factors for use in serum-freeculture methods are purified proteins derived from sera or recombinantproteins derived from recombinant organisms. Use thereof has thefollowing disadvantages: though small in amount, biological componentsare contained; use of an expensive product is required; culture variesamong lots; and the like.

In recent years, serum-free culture methods using protein hydrolysateshave been developed. Such culture methods have similar disadvantages asstated above: a component derived from an organism is contained; costsare high; production varies among different lots; and the like. Thus itis difficult to say that such serum-free culture methods are mostadequate for the production of useful proteins.

In view of the foregoing, there have been demands for a culture methodusing a chemically defined medium that contains as few biologicalcomponents as possible, is inexpensive, causes small variation amonglots, and can result in a boost in protein production. Recently,behavior of glucose and amino acids in a culture solution in fed-batchculture was analyzed, and this analysis revealed that use of anincreased amount of glutamate in fed-batch culture contributed toincrease in an amount of antithrombin that was produced (Non-patentDocument 1). However, this finding is only based on specific CHO cellsexpressing glutamine synthetase, and no demonstration using general CHOcells has been performed. Furthermore, individual effects of other aminoacids have not been investigated. Further, amounts of proteins that areproduced are still not sufficient. Thus there have been strong demandsfor a development of a culture process using a chemically defined mediumthat is capable of offering more enhanced protein production.

-   Non-patent Document 1: Journal of Bioscience and Bioengineering    (2005), 100(5), 502-510

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

The present invention is applicable to a fed-batch culture method usinga medium in which biological medium components are reduced to a minimum,and aims to provide an improved medium so that when cells are cultivatedby fed-batch culture using the medium, the cells produce proteins at ahigh yield.

Means for Solving the Problems

The present inventors extensively and intensively studied to solve theabove problems. Consequently, they found that specific amino acids werefrequently depleted and that maintaining such amino acids at highconcentrations in a serum-free liquid culture enabled cells to produceproteins at a high yield. By this finding, the present invention wascompleted.

Specifically, the present invention provides:

(1) a method of culturing a cell characterized in that a concentrationof serine in a culture solution is maintained at 1 mM or higher at leastduring a certain period after the onset of a cell growth phase, themethod used in a process comprising culturing a cell that is capable ofproducing a desired protein to obtain the desired protein;

(2) the method (1) further characterized in that a concentration oftyrosine in the culture solution is maintained at 1 mM or higher, and/ora concentration of cysteine in the culture solution is maintained at aconcentration of the cysteine in an initial medium or at 0.4 mM orhigher at least during the certain period after the onset of the cellgrowth phase;

(3) a medium for animal cell culture, comprising at least 1 mM of serineor a salt thereof;

(4) a medium for animal cell culture, comprising at least 1 mM of serineor a salt thereof, and further comprising at least 1 mM of tyrosineand/or at least 0.4 mM of cysteine;

(5) a process of producing a desired protein, comprising culturing acell capable of producing the desired protein, by use of the medium (3)or (4) to obtain the desired protein;

(6) a method of culturing a cell, the method used in a processcomprising culturing a cell that is capable of producing a desiredprotein to obtain the desired protein, the method characterized in thata concentration of serine in a culture solution is maintained at 1 mM orhigher at least during a part of or an entire period sufficient toenable the cell that is to be cultured to grow sufficiently, or during apart of or an entire period sufficient to enable adequate production ofthe desired protein that is to be produced;

(7) the method (6) further characterized in that a concentration oftyrosine in the culture solution is maintained at 1 mM or higher, and/ora concentration of cysteine in the culture solution is maintained at aconcentration of the cysteine in an initial medium or at 0.4 mM orhigher at least during a part of or the entire period sufficient toenable the cell that is to be cultured to grow sufficiently, or during apart of or the entire period sufficient to enable adequate production ofthe desired protein that is to be produced;

(8) a method of culturing a cell characterized in that a concentrationof serine in a culture solution is maintained at 1 mM or higher at leastduring a part of or an entire period of an exponential cell growthphase, the method used in a process comprising culturing a cell that iscapable of producing a desired protein to obtain the desired protein;

(9) the method (8) further characterized in that a concentration oftyrosine in the culture solution is maintained at 1 mM or higher, and/ora concentration of cysteine in the culture solution is maintained at aconcentration of the cysteine in an initial medium or higher or at 0.4mM or higher at least during a part of or an entire period of theexponential cell growth phase;

(10) the method (8) or (9), wherein the concentration of serine in theculture solution is maintained at 2 mM or higher at least during a partof or an entire period of the exponential cell growth phase;

(11) a method of culturing a cell characterized in that a concentrationof serine in a culture solution is maintained at 1 mM or higher at leastduring a part of or an entire period from a third day to a tenth day ofthe culture, the method used in a process comprising culturing a cellthat is capable of producing a desired protein to obtain the desiredprotein;

(12) the method (11) further characterized in that a concentration oftyrosine in the culture solution is maintained at 1 mM or higher, and/ora concentration of cysteine in the culture solution is maintained at aconcentration of the cysteine in an initial medium or higher or at 0.4mM or higher at least during a part of or an entire period from thethird day to the tenth day of the culture;

(13) the method (11) or (12), wherein the concentration of serine in theculture solution is maintained at 2 mM or higher at least during a partof or an entire period from the third day to the tenth day of theculture;

(14) any one of the methods or processes (1), (2), and (5)-(13), whereinthe cell is cultured by batch culture, repeated batch culture, fed-batchculture, repeated fed-batch culture, continuous culture, or perfusionculture;

(15) any one of the methods or processes (1), (2), and (5)-(13), whereinthe cell is cultured by fed-batch culture;

(16) the method or process (15), wherein the serine and tyrosine and/orcysteine are fed into the culture solution in multiple batchessequentially or continuously;

(17) any one of the methods or processes (1), (2), and (5)-(16), whereinthe cell is transformed with a gene encoding the desired protein;

(18) the method or process (17), wherein the desired protein is anantibody;

(19) any one of the methods or processes (1), (2), and (5)-(18), whereinthe cell is a mammal cell;

(20) the method (19), wherein the mammal cell is CHO cell;

(21) a fed-batch medium for culturing a cell, comprising serine at aconcentration of 10 mM to 1000 mM;

(22) a fed-batch medium for culturing a cell, comprising serine at aconcentration of 20 mM to 500 mM;

(23) the fed-batch medium (21) or (22), further comprising cysteineand/or tyrosine;

(24) the fed-batch medium (21) or (22), further comprising cysteine andtyrosine;

(25) a method of culturing a cell by fed-batch culture, comprisingadding any one of the fed-batch media (21)-(24);

(26) a process of producing a desired protein by culturing a cell,comprising culturing a cell using any one of the methods (1), (2), and(6)-(20); and

(27) a preparation process of a medicament comprising as an activeingredient a protein produced by the process (5) or (26).

Advantages of the Invention

The present invention can be conveniently used in the production ofphysiologically active peptides or proteins. A feature of the presentinvention is that cultivation using a chemically defined medium freefrom biological components is made more productive of proteins.Furthermore, because the fed-batch medium for use in the presentinvention contains significantly pure amino acids, the mediumcomposition is clearly defined, and the medium is of uniform propertieswith less variations among lots. Use of the medium ensures that aprotein of uniform properties would be obtained as well, thus the mediumis suitable for industrial manufacture. Specifically, the medium greatlycontributes to, for example, mass supply of antibodies for apharmaceutical use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the transition of concentrations of antibodies during theculture period. (Example 1)

FIG. 2 shows the transition of viability during the culture period.(Example 1)

FIG. 3 shows the transition of concentrations of lactate during theculture period. (Example 1)

FIG. 4 shows the transition of concentrations of antibodies during theculture period. (Example 2)

FIG. 5 shows the transition of viability during the culture period.(Example 2)

FIG. 6 shows the transition of concentrations of lactate during theculture period. (Example 2)

FIG. 7 shows the transition of concentrations of antibodies during theculture period. (Example 3)

FIG. 8 shows the transition of concentrations of serine during theculture period. (Example 3)

FIG. 9 shows the transition of concentrations of tyrosine during theculture period. (Example 3)

FIG. 10 shows the amino acid sequence (SEQ ID NO: 2) of a hamstertaurine transporter and the nucleotide sequence (SEQ ID NO: 1) of a geneencoding the same. (Reference Example 1)

FIG. 11 shows a structure of the hamster taurine transporter. (ReferenceExample 1)

FIG. 12 shows a structure of an expression plasmid pHyg/TauT. ReferenceExample 2)

EMBODIMENT OF THE INVENTION

The following specifically describes embodiments of the presentinvention.

A method of the present invention is characterized by comprisingmaintaining, when cells capable of producing desired proteins arecultured to obtain the proteins, serine in a culture solution at a highconcentration. Specifically, the method is characterized by maintainingthe concentration of serine in the culture solution at 1 mM or higher,preferably 2 mM or higher, at least during a certain period of timeafter a cell growth phase has started.

Accordingly, one aspect of the present invention is an animal cellculture medium comprising at least 1 mM of serine or a salt thereof. Inthe present invention, the expression “animal cell culture mediumcomprising at least 1 mM of serine (or a salt thereof)” refers to notonly a medium comprising serine at a concentration of 1 mM or higher inan initial medium but also a medium adjusted such that a concentrationof serine in the culture solution is maintained at 1 mM or higher,preferably 2 mM or higher, by addition of a fed-batch medium or the likeat least during a certain period from or after the onset of the cellgrowth phase.

The method of the present invention is characterized by furthercomprising supplementing the culture solution with tyrosine and/orcysteine, in addition to serine contained at a high concentration.Specifically, the concentration of tyrosine in the culture solution ismaintained at 1 mM or higher and/or the concentration of cysteine ismaintained at a concentration of the cysteine in the initial medium orhigher at least during a certain period from or after the onset of acell growth phase. Since the concentration of cysteine in the initialmedium is normally about 0.4 mM, the concentration of cysteine in theculture solution may be 0.4 mM or higher, preferably 1 mM or higher, atleast during a certain period from or after the start point of the cellgrowth phase, regardless of the concentration of cysteine in the initialmedium.

Accordingly, another aspect of the present invention is an animal cellculture medium comprising 1 mM or higher of serine or a salt thereof,and at least 1 mM or higher of tyrosine and/or 0.4 mM or higher ofcysteine. In the present invention, the expression “animal cell culturemedium comprising at least 1 mM of tyrosine” refers to not only a mediumcomprising tyrosine at a concentration of 1 mM or higher in an initialmedium but also a medium adjusted such that a concentration of tyrosinein the culture solution is maintained at 1 mM or higher by addition of afed-batch medium or the like at least during a certain period from orafter the onset of the cell growth phase. Similarly, the expression“animal cell culture medium comprising at least 0.4 mM of cysteine”refers to not only a medium comprising cysteine at a concentration of0.4 mM or higher in an initial medium but also a medium adjusted suchthat a concentration of cysteine in the culture solution is maintainedat 0.4 mM or higher by addition of a fed-batch medium or the like atleast during a certain period from or after the onset of the cell growthphase.

If the concentration of serine (and tyrosine and/or cysteine) in theculture solution is maintained as described above while cells arecultured, the concentration of serine (and tyrosine and/or cysteine) inthe culture solution is maintained at a predetermined concentration orhigher at least during a part of a period sufficient to enable cellsthat are to be cultured to grow sufficiently, or during a part of aperiod sufficient to enable adequate production of desired proteins thatare to be produced, enabling the cells to produce the proteins at highyields.

Accordingly, another aspect of the present invention is a method ofculturing cells capable of producing desired proteins to obtain theproteins by use of a medium adjusted such that a concentration of serine(and tyrosine and/or cysteine) is maintained at a predeterminedconcentration or higher. Accordingly, another aspect of the presentinvention is a process of producing desired proteins, which process ischaracterized by comprising culturing cells capable of producing thedesired proteins by use of an animal cell culture medium comprising atleast 1 mM of serine or a salt thereof. Another aspect of the presentinvention is a method of producing desired proteins, which method ischaracterized by comprising culturing cells capable of producing thedesired proteins by use of an animal cell culture medium comprising atleast 1 mM of serine or a salt thereof, at least 1 mM of tyrosine,and/or at least 0.4 mM of cysteine.

In the present invention, the period during which the concentration ofserine (and tyrosine and/or cysteine) in the culture solution ismaintained at a predetermined concentration or higher as described abovemay be a part of a period from the start point to the end point of thegrowth phase, or an entire period from the start point to the end pointof the growth phase, or an entire culture period.

The expression “onset (or start point) of a growth phase” as used hereinrefers to a transition period from a lag phase to an accelerated phaseof growth of cultured cells. Thereafter, the phase moves from theaccelerated phase to an exponential growth phase, a decline phase, astationary phase, and then a death phase. (Takeshi Kobayashi andHiroyuki Honda, “Seibutsukagakukogaku” (Biochemical Engineering), TokyoKagaku Dojin, Applied Life Science Series 8, 2002.)

In another aspect of the present invention, the period during which theconcentration of serine (and tyrosine and/or cysteine) in the culturesolution is maintained at a predetermined concentration or higher in theculture method of the present invention may be a part of or an entireperiod sufficient to enable cells that are to be cultured to growsufficiently, or a part of or an entire period sufficient to enableadequate production of desired proteins; specifically, the concentrationmay be maintained at a predetermined concentration or higher during apart of an entire period from the start point to the end point of thegrowth phase of the cells that are cultured. It is especially importantto maintain the concentration of serine (and tyrosine and/or cysteine)in the culture solution at a predetermined concentration or higherduring a certain period from or after the third day of the culture,because an amino acid content of the initial medium is depleted on andafter the third day of the culture. Accordingly, in this aspect of thepresent invention, more specifically, a period in which it is requiredto maintain the concentration of serine (and tyrosine and/or cysteine)in the culture solution at a predetermined concentration or higher maybe at least a part of or an entire period from the third day of theculture to a phase from the decline phase to the stationary phase of thecultured cells, preferably a part of or an entire period from the thirdday of the culture to a phase from the exponential cell growth phase tothe decline phase.

In a further specific aspect, it is preferable to maintain theconcentration of serine (and tyrosine and/or cysteine) in the culturesolution at a predetermined concentration or higher during a period fromat least 4 days after (normally on day 5 of culture) the start point ofthe cell growth phase (normally around day 1 of culture), preferably aperiod from at least 3 days after (normally on day 4 of culture) thestart point of the growth phase, more preferably a period from at least2 days after (normally on day 3 of culture) the start point of thegrowth phase, even more preferably a period from or after the startpoint of the growth phase. Further, it is required to maintain theconcentration of serine (and tyrosine and/or cysteine) in the culturesolution at a predetermined concentration or higher at least until 5days before the end of the culture, preferably until 4 days before theend of the culture, more preferably until 3 days before the end of theculture, in cases in which the culture period is not longer than twoweeks; in cases in which the culture period is longer than two weeks, itis required to maintain the concentration of serine (and tyrosine and/orcysteine) in the culture solution at a predetermined concentration orhigher until the tenth day of the culture, preferably until 5 daysbefore the end of the culture, more preferably until 4 days before theend of the culture, even more preferably until 3 days before the end ofthe culture.

In another aspect of the present invention, it is preferable tocontinuously maintain the concentration of serine (and tyrosine and/orcysteine) in the culture solution at a predetermined concentration orhigher at least during a part of the exponential cell growth phase. Aspecific period is a period from 4 days after the start point of theexponential growth phase, preferably a period from 3 days after thestart point of the exponential growth phase, more preferably a periodfrom the start point of the exponential growth phase. As used herein,the expression “a part of an exponential cell growth phase” refers to apart of the exponential growth phase, an entire period from the startpoint to the end point of the exponential growth phase, or an entireculture period, during which the concentration may be maintained.

In typical cell culture, the start point of the exponential growth phaseis normally around day 3 of culture. Accordingly, in another specificaspect of the present invention, the concentration of serine in theculture solution is maintained at 1 mM or higher, preferably 2 mM orhigher, at least during a part of or an entire culture period from thethird day of the culture. In cases in which the concentrations oftyrosine and/or cysteine are also maintained at predeterminedconcentrations or higher, the concentration of tyrosine in the culturesolution is maintained at 1 mM or higher and/or the concentration ofcysteine is maintained at a concentration of the cysteine in an initialmedium or higher at least during a part of or an entire culture periodfrom the third day of the culture. Since a typical concentration ofcysteine in the initial medium is about 0.4 mM, the said concentrationof cysteine in the culture solution may be 0.4 mM or higher, preferably1 mM or higher, during a part of or an entire culture period from thethird day of the culture, regardless of the concentration of cysteine inthe initial medium.

As used herein, the expression “at least a part of or an entire cultureperiod from the third day of the culture” refers to a culture periodfrom the fourth, fifth, sixth, or seventh day of the culture, or aculture period from the start point of the culture or from the first orsecond day of the culture including a part of or an entire cultureperiod from the third day of the culture.

In the cases in which the culture period is not longer than two weeks,it is preferable to continuously maintain the concentration of serine(and tyrosine and/or cysteine) in the culture solution at apredetermined concentration or higher at least until 5 days before theend of the culture, preferably until 4 days before the end of theculture, more preferably until 3 days before the end of the culture. Inthe cases in which the culture period is longer than two weeks, it ispreferable to continuously maintain the concentration of serine (andtyrosine and/or cysteine) in the culture solution at a predeterminedconcentration or higher at least until the tenth day of the culture,preferably until 5 days before the end of the culture, more preferablyuntil 4 days before the end of the culture, even more preferably until 3days before the end of the culture.

Accordingly, in another aspect of the present invention, theconcentration of serine in the culture solution is maintained at 1 mM orhigher, preferably 2 mM or higher, at least during a part of or anentire period from the third day to the tenth day of the culture. In thecases in which the concentrations of tyrosine and/or cysteine are alsomaintained at predetermined concentrations or higher, the concentrationof tyrosine in the culture solution is maintained at 1 mM or higherand/or the concentration of cysteine is maintained at a concentration ofthe cysteine in an initial medium or higher at least during a part of oran entire period from the third day to the tenth day of the culture.Since the concentration of cysteine in the initial medium is normallyabout 0.4 mM, the concentration of cysteine in the culture solution maybe maintained at 0.4 mM or higher, preferably 1 mM or higher, during apart of or an entire period from the third day to the tenth day of theculture, regardless of the concentration of the cysteine in the initialmedium.

As used herein, the expression “at least a part of or an entire cultureperiod from the third day to the tenth day of the culture” refers to aculture period from the fourth, fifth, sixth, or seventh day of theculture, or a culture period from the start point of the culture or fromthe first or second day of the culture including a part of or an entireculture period from the third day to the tenth day of the culture.

In the above aspect, it is sufficient to maintain the concentration ofserine (and tyrosine and/or cysteine) in the culture solution at apredetermined concentration or higher at least during a part of or anentire period from the third day of the culture. Even when the cultureperiod is shorter than 10 days, if the concentration of serine (andtyrosine and/or cysteine) in the culture solution is maintained at apredetermined concentration or higher during a part of or an entireperiod from the third day of the culture, this is encompassed within thescope of the present invention.

Culturing cells by the method of the present invention enableshigh-yield production of proteins that are bio-product from the cells,and the proteins are isolated from the culture medium and purified toobtain desired proteins.

Culturing cells while maintaining the concentration of serine (andtyrosine and/or cysteine) in the culture solution at a predeterminedconcentration can be achieved either by addition of a high concentrationof serine (and tyrosine and/or cysteine) to the medium at an early stageof the cell culture, or by addition of a medium comprising a highconcentration of serine (and tyrosine and/or cysteine) during theculture to supplement the medium with amino acids.

A preferred timing of commencing supplementing the culture solution withamino acids to maintain the concentration of serine (and tyrosine and/orcysteine) in the culture solution is at least within 3 days from a timewhen the amino acids in the culture solution reach a predeterminedconcentration or lower, preferably within 1 day, most preferably beforethe amino acids reach a predetermined concentration or lower.Supplementation of amino acids may be carried out once only, in butchessequentially, or continuously. Alternatively, the initial medium maycontain a total amount of amino acids required for maintaining the aminoacids at predetermined concentrations.

In general, cell culture methods are classified into batch culture,continuous culture, and fed-batch culture. In the method of the presentinvention, any of these culture methods can be used, but fed-batchculture or continuous culture is preferably used; use of fed-batchculture is especially preferred.

Batch culture is a culture method in which a small amount of seedculture solution is added to a medium and cells are grown without anyaddition of a new medium or discharge of the culture solution during theculture. In the case of using the batch culture in the presentinvention, the medium comprises a high concentration of serine (andtyrosine and/or cysteine) from an initial stage of the cell culture.

Continuous culture is a culture method in which a medium is added anddischarged continuously during the culture. This continuous methodincludes perfusion culture.

Fed-batch culture is also called semi-batch culture because it isbetween batch culture and continuous culture. In the fed-batch culture,a medium is fed continuously or sequentially during cultivation, butunlike the continuous culture, discharge of the culture solution is notcarried out in the culture. The medium to be added in the fed-batchculture (hereinafter “fed-batch medium”) does not necessarily have to bethe same medium as that already used in the culture (hereinafter“initial medium”); namely, a different medium may be added, or onlyspecific components may be added.

As used herein, the term “initial medium” generally refers to a mediumused in an early stage of cell culture. Note that in the case in whichthe fed-batch medium is added in multiple batches, each medium beforethe addition of the fed-batch medium may be referred to as an initialmedium.

In the case of employing the fed-batch culture or continuous culture inthe present invention, the medium that is to be added during the culturemay contain a high concentration of serine (and tyrosine and/orcysteine), or a high concentration of serine (and tyrosine and/orcysteine) may be contained in the culture medium from an initial stageof the cell culture. What is important is to maintain the concentrationof serine, or respective concentrations of serine and tyrosine, orrespective concentrations of serine and cysteine, or respectiveconcentrations of serine, tyrosine, and cysteine, at a predeterminedconcentration or higher at least during a predetermined stage of theculture, as described above. To realize the foregoing, for instance theconcentration of serine (and tyrosine and/or cysteine) in the culturesolution may be monitored to adjust the concentrations of these aminoacids in the medium to be added so that the concentrations of theseamino acids in the culture solution can be controlled. Alternatively, amethod in which, for instance, a stage of a cell growth curve isdetermined on the basis of the number of cells in the culture to controlthe supplementation of the amino acids can be employed.

The following describes in detail serine, tyrosine, and cysteine in theculture solution, by which the present invention is characterized.

Any of serine alone, derivatives thereof, and salts thereof can be used.Natural serine, synthetic serine, or serine produced by generecombination can be used. The concentration of serine in the culturesolution is, for example, 1 mM or higher, preferably 2 mM or higher atleast during a predetermined period in the culture. A conventionallyused medium comprising serine typically comprises about 0.5 mM ofserine; thus it is recognized that the concentration of serine in thepresent invention is significantly high (Dulbecco, R., Freeman, G.Virology 8, p396 (1959), Nature, New Biology (1971) 230, p52)).

Similarly, any of tyrosine alone, derivatives thereof, and salts thereofcan be used. Natural tyrosine, synthetic tyrosine, or tyrosine producedby gene recombination can be used. Similarly, any of cysteine alone,derivatives thereof, and salts thereof can be used, including cystine,which is a dimmer of cysteine. Natural cysteine, synthetic cysteine, orcysteine produced by gene recombination can be used. The concentrationof tyrosine in the culture solution is 1 mM or higher and/or theconcentration of cysteine is at a concentration of the cysteine in theinitial medium or higher at least during a predetermined period duringthe culture.

In the case of employing the fed-batch culture as the method ofculturing cells in the present invention, serine and tyrosine and/orcysteine are dissolved at high concentrations to be enriched in afed-batch medium and a fed-batch medium is added either continuously orsequentially during the culture so that the concentrations of theseamino acids are maintained at predetermined concentrations or higher.Specifically, for example a fed-batch medium comprising L-serine at aconcentration of about 10-1000 mM, preferably 20-500 mM, more preferably50-200 mM, may be used as the fed-batch medium. Alternatively, afed-batch medium comprising L-tyrosine at a concentration of about0.01-1000 mM, preferably 1-200 mM, more preferably 10-100 mM, or afed-batch medium comprising L-cysteine hydrochloride monohydrate at aconcentration of about 0.01-500 mM, preferably 0.1-50 mM, morepreferably 1-10 mM, may be used as the fed-batch medium.

In the case in which the fed-batch medium is added to the culturesolution in the present invention, an amount of the fed-batch medium tobe added sequentially or continuously over a culture period or for acertain period during the culture period may be 1-150%, preferably5-50%, more preferably 8-20%, of an amount of the initial medium.

In the present invention, a period of the addition of the fed-batchmedium to the culture solution includes at least a part of or an entireperiod from the start point to the end point of the cell growth phase ofthe culture. A preferred period is a period from at least 4 days afterthe start point of the exponential growth phase (normally around day 3of the culture) of the cells being cultured, preferably from 3 daysafter the start point of the exponential growth phase, more preferablyfrom the start point of the exponential growth phase (normally aroundday 3 of the culture). It is preferable to start feeding at least within3 days from a time when the concentration of the amino acid in theculture solution reaches a predetermined concentration or lower,preferably within 1 day, most preferably before the concentration of theamino acid reaches the predetermined concentration. In the cases inwhich the culture period is not longer than two weeks, the feeding maybe carried out or continued at least until 5 days before the end of theculture, preferably 4 days before, more preferably 3 days before. In thecases in which the culture period is longer than two weeks, the feedingmay be carried out or continued at least until the tenth day of theculture, preferably until 5 days before the end of the culture, morepreferably until 4 days before the end of the culture, even morepreferably until 3 days before the end of the culture.

Components that are commonly used in media for culturing cells(preferably animal cells) can be appropriately used as other componentsin the culture solution for use in the methods of the present invention,including amino acids, vitamins, lipid factors, energy sources,osmoregulators, iron sources, and pH buffers. In addition to theforegoing components, a minor metal element, surfactant, growthcofactor, nucleoside, or the like may be added. Medium components,including characteristic components, for use in the present inventionmay be divided, and they may be added separately to be used in the cellculture. Specifically, for instance a high concentration of serine aloneand medium components may be used in the cell culture either at the sametime or at different times.

Specific examples of the other components in the culture solutioninclude: amino acids such as L-alanine, L-arginine, L-asparagine,L-aspartic acid, L-glutamine, L-glutamic acid, glycin, L-histidine,L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine,L-phenylalanine, L-proline, L-threonine, L-tryptophan, and L-valine,preferably L-alanine, L-arginine, L-asparagine, L-aspartic acid,L-glutamine, L-glutamic acid, glycin, L-histidine, L-isoleucine,L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline,L-threonine, L-tryptophan, and L-valine; vitamins such as I-inositol,biotin, folic acid, lipoic acid, nicotinamide, nicotinic acid,p-aminobenzoic acid, calcium pantothenate, pyridoxal hydrochloride,pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, and ascorbic acid, preferably biotin, folic acid, lipoic acid,nicotinamide, calcium pantothenate, pyridoxal hydrochloride, riboflavin,thiamine hydrochloride, vitamin B 12, and ascorbic acid; lipid factorssuch as choline chloride, choline tartrate, linoleic acid, oleic acid,and cholesterol, preferably choline chloride; energy sources such asglucose, galactose, mannose, and fructose, preferably glucose;osmoregulators such as sodium chloride, potassium chloride, andpotassium nitrate, preferably sodium chloride; iron sources such as ironEDTA, iron citrate, ferrous chloride, ferric chloride, ferrous sulfate,ferric sulfate, and ferric nitrate, preferably ferric chloride, ironEDTA, and iron citrate; and pH buffers such as sodium bicarbonate,calcium chloride, sodium dihydrogen phosphate, HEPES, and MOPS,preferably sodium bicarbonate.

In addition to the components above, the culture solution can comprise,for example, minor metal elements such as copper sulfate, manganesesulfate, zinc sulfate, magnesium sulfate, nickel chlorid, tin chloride,magnesium chloride, and sodium silicite, preferably copper sulfate, zincsulfate, and magnesium sulfate; surfactants such as Tween 80 andPluronic F68; and growth cofactors such as recombinant insulin,recombinant IGF, recombinant EGF, recombinant FGF, recombinant PDGF,recombinant TGF-alpha, ethanolamine hydrochloride, sodium selenite,retinoic acid, and putrescine hydrochloride, preferably sodium selenite,ethanolamine hydrochloride, recombinant IGF, and putrescinehydrochloride; and nucleosides such as deoxyadenosine, deoxycytidine,deoxyguanosine, adenosine, cytidine, guanosine, and uridine. Inpreferred examples of the present invention described above,antibiotics, such as streptomycin, penicillin G potassium, andgentamicin, and pH indicators, such as phenol red, may be contained.

The amount of other components in the culture solution are normally inthe ranges of suitably 0.05-1500 mg/L amino acid, 0.001-10 mg/Lvitamins, 0-200 mg/L lipid factors, 1-20 g/L energy sources, 0.1-10000mg/L osmoregulators, 0.1-500 mg/L iron source, 1-10000 mg/L pH buffers,0.00001-200 mg/L minor metal elements, 0-5000 mg/L surfactant,0.05-10000 μg/L growth cofactors, and 0.001-50 mg/L nucleosides, but arenot limited to these ranges and can be appropriately determineddepending on the type of the cell cultured, the type of the desiredprotein, and the like.

The pH of the culture solution depends on the cells to be cultured, butnormally is pH 6.8-7.6, and may often suitably be pH 7.0-7.4.

In the present invention, cells can be cultured using a completesynthetic medium in which the foregoing components are dissolved. It isalso possible to use as a basal medium a conventionally known animalcell culture medium, and the medium may be supplemented with thecharacteristic components for use in the present invention. Examples ofcommercially-available basal media that can be used as the animal cellculture medium include: D-MEM (Dulbecco's Modified Eagle Medium),D-MEM/F-12 1:1 Mixture (Dulbecco's Modified Eagle Medium: NutrientMixture F-12), RPMI1640, CHO-S-SFMII (Invitrogen), CHO-SF(Sigma-Aldrich), EX-CELL 301 (JRH biosciences), CD-CHO (Invitrogen), ISCHO-V (Irvine Scientific), and PF-ACF-CHO (Sigma-Aldrich). In cases ofculturing cells by fed-batch culture, such commercially-available mediacan be used as the initial medium at an early stage of the cell culture.The same medium as used as the initial medium may be concentrated andsupplemented with serine and tyrosine and/or cysteine and then used asthe fed-batch medium.

The culture methods of the present invention can be used to culturevarious cells (e.g., bacterial cells, fungal cells, insect cells, plantcells, animal cells) without any limitation. For example, recombinantCOS cells or recombinant CHO cells carrying a gene encoding a desiredprotein prepared by genetic engineering, or fused cells producingantibodies, exemplified by hybridoma such as mouse-human, mouse-mouse,and mouse-rat, can be cultured. The methods of the present invention canbe used to culture animal cells to obtain natural type proteins that theanimal cells produce, or to culture BHK cells, HeLa cells, and the likeas well as the cells described above.

Especially preferred animal cells in the present invention are CHO cellsin to which a gene encoding a desired protein is introduced. The desiredprotein is not particularly limited and may be any protein such asantibodies, such as natural antibodies, antibody fragments, smallantibody fragments or “minibody”, chimeric antibodies, and humanizedantibodies (e.g., anti-IL-6 receptor antibodies, anti-glypican-3antibodies, anti-CD3 antibodies, anti-CD20 antibodies, anti-GPIIb/IIIaantibodies, anti-TNF antibodies, anti-CD25 antibodies, anti-EGFRantibodies, anti-Her2/neu antibodies, anti-RSV antibodies, anti-CD33antibodies, anti-CD52 antibodies, anti-IgE antibodies, anti-CD11aantibodies, anti-VEGF antibodies, anti-VLA4 antibodies) andphysiologically active proteins (e.g., granulocyte colony-stimulatingfactor (G-CSF), granulocyte macrophage colony-stimulating factor(GM-CSF), erythropoietin, interferon, interleukin such as IL-1 and IL-6,t-PA, urokinase, serum albumin, blood coagulation factor), butantibodies are especially preferred.

Antibodies produced by the methods of the present invention include notonly monoclonal antibodies derived from animals, such as human, mouse,rat, hamster, rabbit, and monkey, but also artificially-modified generecombinant antibodies, such as chimeric antibodies, humanizedantibodies, and bispecific antibodies. The immunoglobulin class of anantibody is not particularly limited, and may be any of IgG, such asIgG1, IgG2, IgG3, and IgG4, IgA, IgD, IgE, and IgM, but IgG and IgM arepreferred in pharmaceutical uses. The antibodies of the presentinvention include not only whole antibodies but also antibody fragments,such as Fv, Fab, and F(ab)₂, and minibodies which are single chain Fv(e.g., scFv, sc(Fv)₂) with uni-, bi- or multi-valent binding in whichvariable regions of the antibodies are connected by linkers such aspeptide linkers.

Since culture conditions differ according to types of cells to be used,suitable conditions can be appropriately determined. For example, CHOcells may be cultured, normally, in an atmosphere of CO₂ gas at aconcentration of 0-40%, preferably 2-10%, at 30-39° C., preferably about37° C., for 1-50 days, preferably 1-14 days.

Various bioreactors for animal cell cultures can be used; for example,fermenter-type tank bioreactors, airlift bioreactors, culture flaskbioreactors, spinner flask bioreactors, microcarrier bioreactors,fluidized-bed bioreactors, hollow fiber bioreactors, roller bottlebioreactors, and packed-bed bioreactors.

Culturing cells (preferably animal cells) by the methods of the presentinvention enables high-yield production of proteins.

Some proteins can be produced merely by culturing cells that produce theproteins, while production of some other proteins requires a specialoperation. The operation or conditions may be appropriately determinedaccording to animal cells to be cultured. For example, in the case ofCHO cells transformed by genetic engineering with a vector having a geneencoding a mouse-human chimeric antibody, the cells may be culturedunder the foregoing conditions so that a desired protein is obtained inthe medium within 1-50 days, preferably 5-21 days, more preferably about7-14 days. The resulting protein may be isolated and purified by methodswell known in the art (refer to, for example, Kotaikogakunyumon(Introduction to Antibody Engineering), Chijinshokan Co. Ltd., (1994) p.102-104; Affinity Chromatography Principles & Methods, GE Healthcare,(2003) p. 56-60) to obtain the desired protein.

The present invention enables high-yield production of recombinantantibodies (e.g., natural antibodies, antibody fragments, fragmentedantibodies, chimeric antibodies, humanized antibodies, bispecificantibodies), gene recombinant proteins (e.g., granulocytecolony-stimulating factors (G-CSF), granulocyte macrophagecolony-stimulating factors (GM-CSF), erythropoietins, interferon,interleukin, such as IL-1 and IL-6, t-PA, urokinase, serum albumin,blood coagulation factors), and the like.

In cases in which a protein or polypeptide produced by the methods ofthe present invention (the protein and the polypeptide are sometimesreferred to as the protein of the present invention) has a biologicalactivity that can be utilized as a pharmaceutical, such a protein orpolypeptide may be mixed with a pharmaceutically acceptable carrier oradditive and formulated to produce a medicament. Proteins of the presentinvention, and medicaments comprising as an active ingredient theproteins of the present invention are also encompassed within the scopeof the present invention.

Examples of pharmaceutically acceptable carriers and additives includewater, pharmaceutically acceptable organic solvents, collagen, polyvinylalcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodiumcarboxymethyl cellose, sodium polyacrylate, sodium alginate,water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethyl cellulose, xanthan gum, gum arabic, casein, agar,polyethylene glycol, diglycerol, glycerol, propylene glycol, Vaseline,paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA),mannitol, sorbitol, lactose, and surfactants that are acceptable aspharmaceutical additives.

An actual additive is selected from the foregoing, either alone or incombination, according to a form of a therapeutic agent that is apharmaceutical of the present invention, but is definitely not limitedto those listed above. For example, in cases of using as a formulationfor injection, an additive prepared by dissolving a purified polypeptideinto a solvent, such as physiological saline, a buffer solution, and agrape sugar solution, and adding an adsorption preventive agent, such asTween 80, Tween 20, gelatin, and human serum albumin, may be used. Afreeze-dried additive may be used to realize a form that dissolves andrestructures before use, and for example sugar alcohols and sugars, suchas mannitol and grape sugar, may be used as excipients for freezedrying.

An effective amount of administration of the polypeptide isappropriately selected according to a type of the polypeptide, a type ofdisease to be treated or prevented, an age of a patient, severity ofdisease, and the like. For instance, in cases in which the protein ofthe present invention is an antibody, such as an anti-glypican antibody,the effective amount of administration is selected from the range of0.001 mg to 1000 mg per one kilogram of body weight per administration.The amount of administration can be selected from the range of 0.01 to100000 mg/body of a patient. The amount of administration, however, isnot limited to the foregoing.

Methods of administration of pharmaceuticals of the present inventionmay be either of oral administration and parenteral administration, butparenteral administration is preferred; specific examples includeinjection (e.g., general or local administration by intravenousinjection, intramuscular injection, intraperitoneal injection,subcutaneous injection, or the like), transnasal administration, lungadministration, and percutaneous administration.

EXAMPLES

The present invention is described in detail below, with reference tothe Examples and Reference Examples. The following Examples and the likeillustrate the present invention without, however, limiting the presentinvention thereto.

[Example 1] Fed-Batch Culture Using a Fed-Batch Medium ComprisingSerine, Cysteine, and Tyrosine at High Concentrations and Having a LowpH (a CHO Cell Strain Transformed with an Antibody Gene)

A composition of a medium and a method of preparation are as follows.

Initial medium: A commercially-available animal cell culture medium wasdissolved and then sterilized by filtration.

Fed-batch medium: An animal cell culture medium for use in the initialmedium was dissolved such that a concentration of the animal cellculture medium was three times the concentration of the initial medium.Then, 50 mM of serine, 1.8 mM of cysteine hydrochloride monohydrate, and14.5 mM of tyrosine were added, and a pH was decreased with hydrochloricacid until the medium components were completely dissolved (around pH1.5). After it was confirmed that the medium components were completelydissolved, the resulting medium was sterilized by filtration.

Cells: Humanized IgG (anti-glypican-3 antibody)-producing CHO cellstrain (refer to WO 2006/006693 pamphlet).

The initial medium was charged into a jar-type cell culture apparatus,and the CHO cells were added so as to be 2×10⁵ cells/mL to startcultivation under conditions of 37° C. and 10% CO₂. During a 14-dayculture period, a lower limit of the pH was automatically controlled to7.0, and a concentration of dissolved oxygen was automaticallycontrolled to 40%. From the third day of the culture, the fed-batchmedium was fed at a constant flow rate (1.0 g/hour with respect to 1 Linitial medium), and the cultivation was continued until the fourteenthday. Sampling was carried out at the beginning of the cultivation and onthe third, fifth, seventh, tenth, twelfth, and fourteenth days. Culturesupernatants of the respective samples were subjected to affinitychromatography using protein A to measure concentrations of antibodiesthat were produced, subjected to trypan blue staining to measuresurvival rates, and subjected to immobilized enzyme method to quantitatelactate. As shown in FIG. 1 , in the case of the fed-batch culture(Control) using the fed-batch medium that was not enriched with serine,cysteine, and tyrosine, the concentration of the antibody that wasobtained as a result of the 14-day culture was about 1.6 g/L. On theother hand, in the case of the fed-batch culture (Ser, Cys, Tyr) usingthe fed-batch medium having a low pH and comprising increased amounts ofserine, cysteine, and tyrosine at high concentrations, the concentrationof antibody that was obtained as a result of the 14-day culture wasabout 2.2 g/L; the concentration was higher by about 40%. FIG. 2 showsthe transition of viability (survival rates) during the culture period.Further, as shown in FIG. 3 , the concentration of lactate transitionedat a low level from the third day of the culture in the fed-batchculture using the fed-batch medium having a low pH and enriched withserine, cysteine, and tyrosine, as compared to the fed-batch cultureusing the fed-batch medium that was not enriched with serine, cysteine,and tyrosine.

[Example 2] Fed-Batch Culture Using a Fed-Batch Medium ComprisingSerine, Cysteine, and Tyrosine at High Concentrations and Having a LowpH (a CHO Cell Strain Transformed with an Antibody Gene and HamsterTaurine Transporter Gene)

A composition of a medium and a method of preparation are as follows.

Initial medium: A commercially-available animal cell culture medium wasdissolved and then sterilized by filtration.

Fed-batch medium: An animal cell culture medium for use in the initialmedium was dissolved such that a concentration of the animal cellculture medium was three times the concentration of the initial medium.Then, 50 mM of serine, 1.8 mM of cysteine hydrochloride monohydrate, and14.5 mM of tyrosine were added, and a pH was decreased with hydrochloricacid until the medium components were completely dissolved (around pH1.5). After it was confirmed that the medium components were completelydissolved, the resulting medium was sterilized by filtration.

Cells: Humanized IgG-producing CHO cell strain transformed with ataurine transporter gene.

The initial medium was charged into a jar-type cell culture apparatus,and the CHO cells were added so as to be 2×10⁵ cells/mL to startcultivation under conditions of 37° C. and 10% CO₂. During a 14-dayculture period, a lower limit of the pH was automatically controlled to7.0, and a concentration of dissolved oxygen was automaticallycontrolled to 40%. From the third day of the culture, the fed-batchmedium was fed at a constant flow rate (1.5 g/hour with respect to 1 Linitial medium), and the culture was continued until the fourteenth day.Sampling was carried out at the beginning of the culture and on thethird, fifth, seventh, twelfth, and fourteenth days. Culturesupernatants of the respective samples were subjected to affinitychromatography using protein A to measure concentrations of antibodiesthat were produced, subjected to trypan blue staining to measuresurvival rates, and subjected to immobilized enzyme method to quantitatelactate. As shown in FIG. 4 , in the case of the fed-batch culture(Control) using the fed-batch medium that was not enriched with serine,cysteine, and tyrosine, the concentration of the antibody that wasobtained as a result of the 14-day culture was about 1.3 g/L. On theother hand, in the case of the fed-batch culture (Ser, Cys, Tyr) usingthe fed-batch medium comprising increased amounts of serine, cysteine,and tyrosine at high concentrations and having a low pH, theconcentration of the antibody that was obtained as a result of the14-day culture was about 2.0 g/L; the concentration was higher by about54%. As shown in FIG. 5 , in the case of the fed-batch culture using thefed-batch medium that was not enriched with serine, cysteine, andtyrosine, the survival rate was 52% on the fourteenth day of the 14-dayculture. On the other hand, in the case of the fed-batch culture usingthe fed-batch medium having a low pH and comprising increased amounts ofserine, cysteine, and tyrosine at high concentrations, the survival ratewas 78% on the fourteenth day of the 14-day culture; a high viabilitywas maintained. Further, as shown in FIG. 6 , the concentration oflactate transitioned at a low level from the third day of the culture inthe fed-batch culture using the fed-batch medium having a low pH andenriched with serine, cysteine, and tyrosine, as compared with thefed-batch culture using the fed-batch medium that was not enriched withserine, cysteine, and tyrosine.

[Example 3] Fed-Batch Culture Showing Contribution of HighConcentrations of Serine, Cysteine, and Tyrosine to Achieve High-YieldProduction of Antibodies (a CHO Cell Strain Transformed with an AntibodyGene and a Hamster Taurine Transporter Gene)

A composition of a medium and a method of preparation are as follows.

Initial medium: A commercially-available animal cell culture medium wasdissolved and then sterilized by filtration.

Fed-batch medium (Ser, Cys, Tyr): An animal cell culture medium for usein the initial medium was dissolved such that a concentration of theanimal cell culture medium was three times the concentration of theinitial medium. Then, 50 mM of serine, 1.8 mM of cysteine hydrochloridemonohydrate, and 14.5 mM of tyrosine were added, and a pH was decreasedwith hydrochloric acid until the medium components were completelydissolved (around pH 1.5). After it was confirmed that the mediumcomponents were completely dissolved, the resulting medium wassterilized by filtration.

Fed-batch medium (Ser, Cys): An animal cell culture medium for use inthe initial medium was dissolved such that a concentration of the animalcell culture medium was three times the concentration of the initialmedium. Then, 50 mM of serine and 1.8 mM of cysteine hydrochloridemonohydrate were added, and a pH was decreased with hydrochloric aciduntil the medium components were completely dissolved (around pH 1.0).After it was confirmed that the medium components were completelydissolved, the resulting medium was sterilized by filtration.

Fed-batch medium (Ser, Tyr): An animal cell culture medium for use inthe initial medium was dissolved such that a concentration of the animalcell culture medium was three times the concentration of the initialmedium. Then, 50 mM of serine and 14.5 mM of tyrosine were added, and apH was decreased with hydrochloric acid until the medium components werecompletely dissolved (around pH 1.0). After it was confirmed that themedium components were completely dissolved, the resulting medium wassterilized by filtration.

Fed-batch medium (Cys, Tyr): An animal cell culture medium for use inthe initial medium was dissolved such that a concentration of the animalcell culture medium was three times the concentration of the initialmedium. Then, 1.8 mM of cysteine hydrochloride monohydrate and 14.5 mMof tyrosine were added, and a pH was decreased with hydrochloric aciduntil the medium components were completely dissolved (around pH 1.0).After it was confirmed that the medium components were completelydissolved, the resulting medium was sterilized by filtration.

Cells: Humanized IgG-producing CHO cell strain transformed with ataurine transporter gene.

The initial medium was charged into a jar-type cell culture apparatus,and the CHO cells were added so as to be 2×10⁵ cells/mL to startcultivation under conditions of 37° C. and 10% CO₂. During a 14-dayculture period, a lower limit of the pH was automatically controlled to7.0, and a concentration of dissolved oxygen was automaticallycontrolled to 40%. From the third day of the culture, the fed-batchmedium was fed at a constant flow rate (1.0 g/hour with respect to 1 Linitial medium), and the culture was continued until the fourteenth day.Sampling was carried out at the beginning of the culture and on thethird, fifth, seventh, tenth, twelfth, and fourteenth days.Concentrations of antibodies that were produced in culture supernatantsof the respective samples were measured by affinity chromatography usingprotein A, and concentrations of amino acids, serine and tyrosine, inthe culture supernatants of the respective samples were measured byamino acid analysis using an ion exchange column. As shown in FIG. 7 ,in the case of the fed-batch culture (Control) using the fed-batchmedium that was not enriched with serine, cysteine, and tyrosine, theconcentration of the antibody that was obtained as a result of the14-day culture was about 1.16 g/L. On the other hand, in the case of thefed-batch culture (Ser, Cys, Tyr) using the fed-batch medium having alow pH and comprising increased amounts of serine, cysteine, andtyrosine at high concentrations, the concentration of the antibody thatwas obtained as a result of the 14-day culture was 1.87 g/L; in the caseof the fed-batch culture (Ser, Cys) using the fed-batch medium having alow pH and comprising increased amounts of serine and cysteine at highconcentrations, the concentration of the antibody that was obtained as aresult of the 14-day culture was 1.47 g/L; in the case of the fed-batchculture (Ser, Tyr) using the fed-batch medium having a low pH andcomprising increased amounts of serine and tyrosine at highconcentrations, the concentration of the antibody that was obtained as aresult of the 14-day culture was 1.41 g/L; and in the case of thefed-batch culture (Cys, Tyr) using the fed-batch medium having a low pHand comprising increased amounts of cysteine and tyrosine, theconcentration of the antibody that was obtained as a result of the14-day culture was 1.18 g/L. The results suggest that highconcentrations of serine, cysteine, and tyrosine contributed in thisorder to high-yield production of antibodies. The results also suggestthat addition of high concentrations of serine, cysteine, and tyrosineall together to the fed-batch medium in the same instance mostsignificantly contributed to high-yield production of antibodies.

FIGS. 8 and 9 show the transitions of the concentrations of serine andtyrosine during the 14-day culture period. In the control, theconcentration of serine was 0.63 mM and the concentration of tyrosinewas 0.34 mM on the fifth day, and the concentration of serine was 0.4 mMor lower and the concentration of tyrosine was 0.4 mM or lower from theseventh day of the culture. On the other hand, the concentration ofserine was maintained at 2 mM or higher in the culture using thefed-batch medium supplemented with serine, and the concentration oftyrosine was maintained at 1 mM or higher in the culture using thefed-batch medium supplemented with tyrosine.

The following Reference Examples describe the preparation of humanizedIgG-producing CHO cell strains transformed with a taurine transportergene that were used in Examples 2 and 3 described above.

[Reference Example 1] Cloning of a Hamster Taurine Transporter GeneDerived from CHO Cells

Total RNA was extracted from cells producing anti-IL-6 receptor antibody(JP H08-99902 A), obtained by transforming an anti-IL-6 receptorantibody gene into CHO DXB11 cells. Thereafter, polyA-dependent cDNA wassynthesized. A hamster taurine transporter (Hamster TauT) gene wasobtained by PCR using as a template cDNA fragmented by three types ofrestriction enzymes, SalI, XhoI, and EcoRI. A PCR primers containing 5′,3′ conservative sequences which are common between known Rat/Mouse TauTwere designed and used. After nucleotide sequence of the cloned gene wasdetermined, it was confirmed that the gene encoded Hamster TauT, basedon homology with known TauT from other biological species (FIG. 10 ).The amino acid sequence of Hamster TauT was highly homologeous with thatfrom Mouse (96% Identity), Rat (96% Identity), and Human (93% Identity);therefore, it was speculated that Hamster TauT was a transporter having12 trans-membrane domains (FIG. 11 ).

[Reference Example 2] Preparation of CHO Cell Strains into which aHamster Taurine Transporter Gene is Introduced

An expression plasmid pHyg/TauT (FIG. 12 ) with CMV promoter wasconstructed by adding a Kozak sequence to the Hamster TauT gene(hereinafter “TauT”) obtained by the cloning in Reference Example 1. CHOcells expressing anti-glypican-3 antibody, as the parent strain, (referto WO 2006/006693 pamphlet), were transformed by electroporation withthe plasmid pHyg/TauT or a control plasmid pHyg without TauT. Cellstransformed with the expression plasmid were selected in the presence ofHygromycin (400 μg/ml), followed by expansion of all cell strainsshowing stable growth (pHyg/TauT: 8 strains, pHyg: 7 strains). AfterTauT mRNA was prepared, the TaqMan procedure was performed to select 7strains showing superior expression over the parent strain to obtaincells transformed with pHyg/TauT. An average amount of mRNA expressionof the transformed cells (7 strains) was about 40-fold the control (7strains).

Free Text of Sequence List

<SEQ ID NO: 1> SEQ ID NO: 1 shows the base sequence of a gene encoding ahamster taurine transporter.

<SEQ ID NO: 2> SEQ ID NO: 2 shows the amino acid sequence of a hamstertaurine transporter.

1. A method of culturing a cell comprising culturing a cell capable ofproducing a desired protein in a culture solution in which aconcentration of serine in the culture solution is maintained at 1 mM orhigher at least during a certain period after onset of a cell growthphase, and obtaining the desired protein.
 2. The method of claim 1,wherein a concentration of tyrosine in the culture solution ismaintained at 1 mM or higher, and/or a concentration of cysteine in theculture solution is maintained at a concentration of the cysteine in aninitial medium or at 0.4 mM or higher at least during the certain periodafter onset of the cell growth phase.
 3. A method of culturing a cell,comprising culturing a cell capable of producing a desired protein aculture solution in which a concentration of serine in the culturesolution is maintained at 1 mM or higher at least during a part of or anentire period sufficient to enable the cell that is to be cultured togrow sufficiently, or during a part of or an entire period sufficient toenable adequate production of the desired protein that is to beproduced.
 4. The method of claim 3, wherein a concentration of tyrosinein the culture solution is maintained at 1 mM or higher, and/or aconcentration of cysteine in the culture solution is maintained at aconcentration of the cysteine in an initial medium or at 0.4 mM orhigher at least during a part of or the entire period sufficient toenable the cell that is to be cultured to grow sufficiently, or during apart of or the entire period sufficient to enable adequate production ofthe desired protein that is to be produced.
 5. A method of culturing acell, comprising culturing a cell capable of producing a desired proteinin a culture solution in which a concentration of serine in the culturesolution is maintained at 1 mM or higher at least during a part of or anentire period of an exponential cell growth phase, and obtaining thedesired protein.
 6. The method of claim 5, wherein a concentration oftyrosine in the culture solution is maintained at 1 mM or higher, and/ora concentration of cysteine in the culture solution is maintained at aconcentration of the cysteine in an initial medium or higher or at 0.4mM or higher at least during a part of or an entire period of theexponential cell growth phase.
 7. The method of claim 4 wherein theconcentration of serine in the culture solution is maintained at 2 mM orhigher at least during a part of or an entire period of the exponentialcell growth phase.
 8. A method of culturing a cell, comprising culturinga cell capable of producing a desired protein in a culture solution inwhich a concentration of serine in the culture solution is maintained at1 mM or higher at least during a part of or an entire period from athird day to a tenth day of the culture, and obtaining the desiredprotein.
 9. The method of claim 7, wherein a concentration of tyrosinein the culture solution is maintained at 1 mM or higher, and/or aconcentration of cysteine in the culture solution is maintained at aconcentration of the cysteine in an initial medium or higher or at 0.4mM or higher at least during a part of or an entire period from thethird day to the tenth day of the culture.
 10. The method of claim 8,wherein the concentration of serine in the culture solution ismaintained at 2 mM or higher at least during a part of or an entireperiod from the third day to the tenth day of the culture.
 11. Themethod of claim 1, wherein the cell is cultured by batch culture,repeated batch culture, fed-batch culture, repeated fed-batch culture,continuous culture, or perfusion culture.
 12. The method of claim 1,wherein the cell is cultured by fed-batch culture.
 13. The method ofclaim 12, wherein the serine and tyrosine and/or cysteine are fed intothe culture solution in multiple batches sequentially or continuously.14. The method of claim 1, wherein the cell is transformed with a geneencoding the desired protein.
 15. The method of claim 14, wherein thedesired protein is an antibody.
 16. The method of claim 1, wherein thecell is a mammal cell.
 17. The method of claim 16, wherein the mammalcell is a CHO cell.
 18. A fed-batch medium for culturing a cell,comprising serine at a concentration of 10 mM to 1000 mM.
 19. Afed-batch medium for culturing a cell, comprising serine at aconcentration of 20 mM to 500 mM.
 20. The fed-batch medium of claim 18,further comprising cysteine and/or tyrosine.
 21. The fed-batch medium ofclaim 18, further comprising cysteine and tyrosine.